Stem Cell Populations and Methods of Use

ABSTRACT

Populations of stem cells and methods for their isolation and use are provided. These stem cell populations comprise aldehyde dehydrogenase positive (ALDH br ) cells isolated from bone marrow, and ALDH br  CD105 +  cells derived from any stem cell source. These populations may also comprise cells expressing such surface markers as CD34, CD38, CD41, CD45, CD105, CD133, CD135, CD117, and HLA-DR, and/or are substantially free from such cell surface markers as CD3, CD7, CD 10, CD 13, CD 14, C1319, CD33, CD35, CD56, CD 127, CD 138, and glycophorin A. The population may also comprise cells expressing CD90. The stem cell populations of the invention are isolated from a stem cell source such as bone marrow, peripheral blood, umbilical cord blood, and fetal liver. Methods of the invention comprise isolating and purifying stem cell populations from stem cell sources, and methods of using these cells to reconstitute, repair, and regenerate tissues.

FIELD OF THE INVENTION

The present invention relates to populations of stem cells, methods forisolating these stem cell populations, and methods of reconstituting,repairing, and regenerating tissue using the same. The inventionadditionally relates to methods of screening agents that promote growth,engraftment, and differentiation of stem cells.

BACKGROUND OF THE INVENTION

Stem and progenitor cells (SPC) reproduce and maintain developmentalpotential until specific biological signals induce the cells todifferentiate into a specific cell type or tissue type. Adult stem andprogenitor cells (ASPC) are small populations of SPC that remain intissues of an organism following birth and are continuously renewedduring a lifetime. In vitro colony assays have demonstrated that bonemarrow (BM), mobilized peripheral blood (MPB), and umbilical cord blood(UCB), all contain a variety of ASPC. Bone marrow is particularly richin multipotential ASPC.

ASPC populations that give rise to a lineage are likely to beheterogeneous. Thus, not all stem cells are CD34⁺. Similarly, while manycommon lymphoid progenitors are CD7⁺ and CD3⁻, some are CD34⁺ and othersare CD34⁻. In part, such heterogeneity may reflect the fact that cellsurface antigen expression, including CD34 expression, can depend oncell cycle or activation as well as developmental potential.

Some studies have suggested that the most primitive human hematopoieticstem cells (HSC) express the CD34 surface marker (i.e., are CD34⁺), lackobvious lineage commitment markers (designated Lin⁻), and express low toundetectable levels of other cell surface markers including CD38, CD71,CD45RA, and Thy-1. See, for example, Terstaypen et al. (1991) Blood77:1218; Landsdorp et al. (1993) J. Exp. Med. 178:787; Cicuttini et al.(1994) Growth Factors 10: 127; De Bruyn et al. (1995) Stem Cells 13:281;Di Giusto et al. (1994) Blood 84:421 (1994); Hao et al. (1995) Blood86:374; Huang et al. (1994) Blood 83:1515; Muench et al. (1994) Blood83:3170; and Rusten et al. (1994) Blood 84:1473. Conversely, otherstudies using long-term murine bone marrow transplant models haveindicated that CD34^(lo/−) cells contain a hematopoietic stem cellpopulation that is capable of durably generating lymphoid and myeloidlineages following transplantation. See, for example, Osawa et al.(1996) Science 273:242; Morel et al. (1996) Blood 88:629a; and Jones etal. (1996) Blood 88:487, in which a population of small Lin⁻ CD34^(lo/−)ALDH⁺ cells capable of durably generating lymphoid and myeloid lineagesfollowing engraftment was identified.

Some evidence also suggests that ASPC that appear to be committed to acertain cell lineage, such as blood cells, may retain the ability toform additional tissues, such as muscle or nerves, under appropriateconditions. For example, CD133⁺ mesenchymal stem-like cells generallyexpress more neuronal cell markers than CD34⁺/CD133⁻ cells (Padovan etal (2003) Cell Transplantation 12:839). Additionally, SPC becomeprogressively restricted in their developmental potential. Thus, humanstem cell populations that express CD45 and CD34, but not CD38, arehighly enriched for multipotential (pluripotent) hematopoietic stem andprogenitor cells; whereas, cells that express CD45, CD34, and CD38concomitantly are more restricted developmentally. Similarly, human stemcells that generate endothelial, but not hematopoietic, colonies invitro generally express CD31, but not CD45 or CD34. Mesenchymal stemcells generally express CD105 and CD135. Chen et al. (2002) Proc. Natl.Acad. Sci. USA. 99:15468; Chen et al. (2003) Immunity 19:525; Pierelliet al (2001) Leuk. Lymphoma. 42:1195. Morita et al. (2003) Eur. J.Haematol. 71:351.

Aldehyde dehydrogenase (ALDH) is a marker that can be used to enrichAPSC. See, U.S. Pat. No. 6,537,807, herein incorporated by reference inits entirety. A fluorescent ALDH reaction product must be used toidentify cells via flow cytometry because the marker is not expressed onthe cell surface. See, for example, U.S. Pat. No. 6,627,759, hereinincorporated by reference in its entirety. Expression of ALDH iselevated significantly in hematopoietic, neural, and potentially othertypes of ASPC. Cells can be further enriched by gating on lowgranularity, i.e., side scatter channel (SSC^(lo)) cells. ALDH-positivecells do not co-segregate with CD34. CD34⁺ cell populations includeALDH^(bright) (ALDH^(br)) and ALDH^(dim) cells that, respectively,express high and low levels of enzyme (Storms et al. (1999) Proc. Natl.Acad. Sci. USA 96:9118). The ALDH^(br) cells include virtually all ofthe stem cells, as evidenced by this cell population's ability togenerate multipotential cell colonies in vitro, its ability toreconstitute NOD-SCID mice over a long term, and its ability to rapidlyhome to the bone marrow in NOD-SCID mice. Conversely, the ALDH^(dim)populations, despite being CD34⁺ cells, have very limited colony-formingability, fail to home effectively, and only generate short-termreconstitution in NOD-SCID animals. Thus, ALDH expression can be used todistinguish and isolate functionally active from functionally inactiveASPC CD34⁺ cells. Heterogeneity in umbilical cord blood (UCB) ALDH^(br)populations with regard to CD45 and CD31 expression has also beenreported (Hess et al. (2003) Blood, 102:383 A).

Technologies for isolating and preparing therapeutically active ASPCfrom bone marrow (BM) or mobilized peripheral blood (MPB) areparticularly useful because the patient only needs minimally invasiveprocedures for stem cell therapy. Moreover, because ASPC populations areautologous, the grafts will not be subject to rejection. Allogeneic ASPCpopulations derived from the BM, MPB, or from umbilical cord blood (UCB)of graft donors are also useful. However, to prevent graft rejection,histocompatible donors and immunosuppressive protocols that do notinterfere with graft function are needed.

The therapeutic utility of ASPC is well established, and, while notbeing bound by any mechanism of action or theory, considerable evidenceexists that ASPC cell populations can also generate non-hematopoietictissues in transplant recipients. See, for example, Verfaillie (2002)Trends in Cell Biol. 12:502; Ferrari et al. (1998) Science 279:1528;Gussoni et al. (1999) Nature 401:390-394; Orlic et al. (2001) Nature410:701-705; Jackson et al. (2001) J. Clin. Invest. 107:1395-1402; Grantet al. (2002) Nat. Med. 8:607-602; Mezey et al. (2001) Science290:1779-1782; Brazelton et al. (2000) Science 290:1775-1779; Krause etal. (2001) Cell 105:369-377; Petersen et al. (1999) Science284:1168-1170; Lagasse et al. (2000) Nat. Med. 6:1229-1234; Rehman etal. (2003) Circulation 107:1164-1169. In addition, some evidencesuggests that transplanted ASPC induce host stem cells to repair tissues(Verfaillie (2002) Trends in Cell Biol. 12:502). However, because cellpopulations from stem cell sources contain only a small percentage ofASPC, there is a need for methods of identifying the functional minorityversus the non-functional majority. Once these cells are identified,therapies can be improved using custom-engineered grafts that not onlycontain all necessary cells for a therapeutic result, but also lackpotentially dangerous, contaminating cells. Moreover, identificationmethods allow the useful ASPC to be concentrated, which reduces theamount of material that must be transplanted, thereby reducing tissuedamage and toxicity and increasing efficacy. In addition, such selectedcells can be used to generate mesenchymal cells that can be used torepair or replace tissues such as nerves, muscles, and endothelium. Stemcells can also be propagated in vitro and expanded into mesenchymal andhematopoietic cell lines to further increase the number of SPC or tissuecells that can be used for transplantation.

SUMMARY OF THE INVENTION

Populations of stem cells and methods for their isolation and use areprovided. These stem cell populations comprise aldehyde dehydrogenasepositive (ALDH^(br)) cells isolated from bone marrow, and ALDH^(br)CD105⁺ cells derived from any stem cell source. These populations mayalso comprise cells expressing such surface markers as CD34, CD38, CD41,CD45, CD105, CD133, CD135, CD117, and HLA-DR, and/or are substantiallyfree from such cell surface markers as CD3, CD7, CD10, CD13, CD14, CD19,CD33, CD35, CD56, CD127, CD138, and glycophorin A. The population mayalso comprise cells expressing CD90. The stem cell populations of theinvention are isolated from a stem cell source such as bone marrow,peripheral blood, umbilical cord blood, and fetal liver.

Methods of the invention comprise isolating and purifying stem cellpopulations from stem cell sources, and methods of using these cells toreconstitute, repair, and regenerate tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the scatterplots from Example 1 for the isotype andfluorescent controls.

FIG. 2 shows the expression of surface markers from Example 1.

FIG. 3 shows a gated scatterplot from Example 1 of the expression ofALDH^(br)SSC^(lo) in different grafts.

FIG. 4 shows the expression of ALDH^(br)SSC^(lo), cell populations indifferent grafts for each test subject from Example 1.

FIG. 5 shows the scatterplots from Example 1 for ALDH^(br) andALDH_(dim) cells from bone marrow, umbilical cord blood, and mobilizedperipheral blood are shown for cells expressing CD34, CD105, CD133, andnegative for expression of CD45.

FIG. 6 shows a comparison of CD34⁺ALDH^(br) subpopulations from Example1.

FIG. 7 shows the results of hematopoietic progenitor colony formingassays for four bone marrow samples cultured for two weeks. Filled barsare data for marrow samples following removal of erythrocytes; open barsare cell suspensions following reaction with the ALDH substrate butprior to sorting; and hatched bars are enriched ALDH^(br)SSC^(lo)populations after staining.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to populations of stem cells, methodsfor isolating these stem cell populations, and methods of using thesestem cell populations to reconstitute, regenerate, or repair tissue.These stem cell populations comprise stem and progenitor cells (SPC)that are ALDH^(br), and, thus, contain most or all of the stem cellspresent in a stem cell source. The ALDH^(br) cell population furthercomprises subpopulations of cells that express combinations of cellmarkers, such as CD105, or have low granularity (as measured by flowcytometry), that serve to identify those subpopulations useful forparticular embodiments of the invention such as for reconstituting,regenerating, or repairing a disease of interest or for manufacturingkits.

Using a combination of cell surface markers and others markers such asintracellular enzymes and the light scattering properties of the cells,stem cell grafts of the invention can be advantageously “tailored” forparticular therapeutic uses. Also, because some of these unique stemcell populations represent certain cell lineages, the populations can beused to both selectively reconstitute certain cell lineages in vivo.Stem cells that give rise to hematopoietic lineages can be used toincrease the concentration and potency of stem cell grafts and, thereby,decrease toxicity. Advantageously, these cells can be sorted fromautologous bone marrow and peripheral blood, thus further reducing thechance of rejection and increasing the efficacy of stem cell grafts.Stem cells that give rise to mesenchymal tissues such as bone, nerves,oligodendrocytes, muscles, vasculature, bone marrow stroma, and dermiscan be used to repair or replace diseased or damaged tissues. Thus, thenovel combination of CD markers disclosed herein confers such additionaladvantages as identification of the stem cell sources that arefunctionally and quantitatively best for use in isolating cells for stemcell grafts. By “isolated” is intended stem cells collected from amammal and contacted with a cell marker, including but not limited to anantibody (conjugated or unconjugated), a fluorescent marker, anenzymatic marker, a dye, a stain, and the like.

By “cell surface marker” is intended a protein expressed on the surfaceof a cell, which is detectable via specific antibodies. Cell surfacemarkers that are useful in the invention include, but are not limitedto, the CD (clusters of differentiation) antigens CD1a, CD2, CD3, CD5,CD7, CD8, CD10, CD13, CD14, CD16, CD19, CD29, CD31, CD33, CD34, CD35,CD38, CD41, CD45, CD56, CD71, CD73, CD90, CD105, CD115, CD117, CD124,CD127, CD130, CD138, CD144, CD166, HLA-A, HLA-B, HLA-C, HLA-DR, VEGFreceptor 1 (VEGF-R1), VEGF receptor-2 (VEGF-R2), and glycophorin A. By“intracellular marker” is intended expression of a gene or gene productsuch as an enzyme that is detectable. For example, aldehydedehydrogenase (ALDH) is an intracellular enzyme that is expressed inmost hematopoietic stem cells. It can be detected via flow cytometry byusing fluorescent substrates.

Populations may be further analyzed based on light scattering propertiesof the cells based on side scatter channel (SSC) brightness and forwardscatter channel (FSC) brightness. By “side scatter” is intended theamount of light scattered orthogonally (about 90° from the direction ofthe laser source), as measured by flow cytometry. By “forward scatter”is intended the amount of light scattered generally less than 90° fromthe direction of the light source. Generally, as cell granularityincreases, the side scatter increases and as cell diameter increases,the forward scatter increases. Side scatter and forward scatter aremeasured as intensity of light. Those skilled in the art recognize thatthe amount of side scatter can be differentiated using user-definedsettings. By the terms “low side scatter” and “SSC^(lo)” is intendedless than about 50% intensity, less than about 40% intensity, less thanabout 30% intensity, or even less intensity, in the side scatter channelof the flow cytometer. Conversely, “high side scatter” or “SSC^(hi)”cells are the reciprocal population of cells that are not SSC^(lo).Forward scatter is defined in the same manner as side scatter but thelight is collected in forward scatter channel. Thus, the embodiments ofthe invention include selection of stem cell populations based oncombinations of cell surface markers, intracellular markers, and thelight scattering properties of cells obtained from a stem cell source.

The populations of stem cells disclosed herein can comprise ALDH^(br)cells that can be sorted based on the positive expression of markers. By“positive for expression” is intended the marker of interest, whetherintracellular or extracellular, is detectable in or on a cell using anymethod, including, but not limited to, flow cytometry. The terms“positive for expression,” “positively expressing,” “expressing,” “⁺”used in superscript, and “^(pos)” used in superscript are usedinterchangeably herein. By “negative for expression” is intended themarker of interest, whether intracellular or extracellular, is notdetectable in or on a cell using any method, including but not limitedto flow cytometry. The terms “negative for expression,” “negativeexpressing”, “not expressing,” “⁻” used in superscript, and “^(neg)”used in superscript are used interchangeably herein.

By “^(br)” used in superscript is intended positive expression of amarker of interest that is brighter as measured by fluorescence (usingfor example FACS) than other cells also positive for expression. Thoseskilled in the art recognize that brightness is based on a threshold ofdetection. Generally, one of skill in the art will analyze the negativecontrol tube first, and set a gate (bitmap) around the population ofinterest by FSC and SSC and adjust the photomultiplier tube voltages andgains for fluorescence in the desired emission wavelengths, such that97% of the cells appear unstained for the fluorescence marker with thenegative control. Once these parameters are established, stained cellsare analyzed and fluorescence recorded as relative to the unstainedfluorescent cell population. As used herein the term “bright” or “^(br)”in superscript is intended greater than about 20-fold, greater thanabout 30-fold, greater than about 40-fold, greater than about 50-fold,greater than about 60-fold, greater than about 70-fold, greater thanabout 80-fold, greater than about 90-fold, greater than about 100-fold,or more, increase in detectable fluorescence relative to unstainedcontrol cells. Conversely, as used herein, the terms “dim” or “^(dim)”in superscript is intended the reciprocal population of those defined as“bright” or “^(br)”.

In some embodiments, cells within a population of interest expressmarkers such as CD29, the integrin β1 subunit expressed on most cells;CD31, a homotypic adhesion molecule found on all endothelial cells andsome platelets and leukocytes; CD34, a highly glycosylated type Itransmembrane protein expressed on 1-4% of bone marrow cells; CD38, atype II transmembrane protein found on immature T and B cells but notmost mature peripheral lymphocytes; CD41, the integrin αIIb subunit thatis expressed on platelets and megakaryocytes; CD45, the leukocyte commonantigen found on all cells of hematopoietic origin; CD73, anecto-5′-nucelotidase differentially expressed on subsets of maturelymphocytes and some endothelial and epithelial cells; CD90, a GPI-cellanchored molecule found on prothymocyte cells in humans; CD105, adisulfide-linked homodimer found on endothelial cells but absent frommost T and B cells; CD115, the macrophage colony stimulating factorreceptor expressed primarily on cells of the mononuclear-phagocyticlineage; CD117, the c-kit ligand receptor found on 1-4% of bone marrowstem cells; CD133, a pentaspan transmembrane glycoprotein expressed onprimitive hematopoietic progenitor cells; CD135, the Flt3 ligandreceptor that is expressed on CD34⁺ hematopoietic stem cells; CD138, anextracellular matrix receptor expressed on immature B cells and plasmacells; CD144, the VE-cadherin molecule that organizes adheres junctionin endothelial cells; CD166, found on thymic epithelium, activated Tcells, and neurons; HLA-A, HLA-B, HLA-C, forms of the MHC Class Imolecule; HLA-DR, the MHC Class II molecule; the VEGF receptors 1 and 2,found on hematopoietic stem cells and vascular endothelium; and anycombinations of these markers.

In one embodiment, the population of cells comprises ALDH^(br) cellswherein at least 10% to 100% of the cells express at least CD41, atleast CD105, or at least HLA-DR. In another embodiment, the populationof cells comprises ALDH^(br) SSC^(lo) cells wherein at least 10% to 100%of the cells express any combination of CD41, CD105, and HLA-DR.

In some embodiments, the lack of expression of a cell surface markerdefines stem cell populations of the invention. Examples includepopulations of stem cells comprising ALDH^(br) SSC^(lo) cellssubstantially free of cells expressing the following markers: CD1a, alymphoid marker structurally similar to MHC Class I; CD2, a pan lymphoidmarker associated with antigen recognition; CD3, a member of the T cellreceptor complex; CD5, expressed on mature T and B cells; CD7, an earlyT cell lineage marker; CD10, a type II membrane metalloproteaseexpressed on early T and B cell precursors; CD13, a type II membranemetalloprotease expressed on granulocytes monocytes and theirprecursors; CD14, a GPI-linked protein expressed mainly onmyelomonocytic lineage cells; CD19, a component of the B cell antigensignaling complex; CD33, a sialic acid binding protein that is absentfrom pluripotent stem cells but appears on myelomonocytic precursorsafter CD34; CD35, the complement receptor type 1, which is expressed onmany lymphoid and myelomonocytic cells; CD56, an isoform of the neuraladhesion molecule found exclusively on natural killer (NK) cells; CD127,the high affinity interleukin 7 receptor expressed on lymphocytes;CD138, an extracellular matrix receptor found on immature B cells andplasma cells; glycophorin A, asialoglycoprotein present on human redblood cells and embryoid precursors; and any combinations of thesemarkers. In some embodiments stem cells are Lin⁻; these stem cells donot yet express lineage-commitment cell surface markers, and thereforecomprise a greater number of hematopoietic stem cells. By the term“Lin⁻” is intended that the cell lacks expression of the cell surfacemarkers CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, CD14, andglycophorin A (gly A).

Based on these unique cell surface marker signatures, individual stemcell populations having unique functional characteristics have beenidentified.

In some embodiments at least 10%, 20%, or 30% of the ALDH^(br) cellswithin a stem cell population of the invention express the cell markersof interest; in other embodiments at least 40%, 50%, or 60% of theALDH^(br) cells within the stem cell population express the cell markersof interest; in yet other embodiments at least 70%, 80%, or 90% of theALDH^(br) cells within the stem cell population express the cell markersof interest; in still other embodiments at least 95%, 96%, 97%, 98%,99%, or even 100% of the ALDH^(br) cells within the stem cell populationexpress the cell markers of interest. By “substantially free” isintended less than about 5%, 4%, 3%, 2%, 1%, or even 0% of the cells inthe population express the marker of interest. While the isolation ofpurified cell population from bone marrow is specifically exemplifiedherein, the isolation of such cells from other sources, includingumbilical cord blood, peripheral blood, and fetal liver, is alsocontemplated.

Selective methods known in the art and described herein can be used tofurther characterize SPC, including ASPC. Commonly, sources of SPC andASPC are reacted with monoclonal antibodies, and subpopulations of cellsexpressing cognate cell surface antigens are either positively ornegatively selected with immunomagnetic beads by complement mediatedlysis, agglutination methods, or fluorescence activated cell sorting(FACS). The functional attributes of the resulting subpopulations with adefined cell surface phenotype are then determined using acolony-forming assay. Once the phenotype of cells that do and do nothave SPC or ASPC activity is known, these methods can be used to selectappropriate SPC or ASPC for therapeutic transplantation.

If desired, a large proportion of terminally differentiated cells may beremoved by initially using a negative selection separation step. Forexample, magnetic bead separations may be used initially to remove largenumbers of lineage committed cells. Desirably, at least about 80%,usually at least about 70% of the total cells, will be removed.

Procedures for cell separation may include, but are not limited to,positive or negative selection by means of magnetic separation usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody or used in conjunction with amonoclonal antibody, including, but not limited to, complement andcytotoxins, and “panning” with antibody attached to a solid matrix,e.g., plate, elutriation, or any other convenient technique.

Techniques providing accurate cell separation include, but are notlimited to, flow cytometry, which can have varying degrees ofsophistication, e.g., a plurality of color channels, low angle andobtuse light scattering detecting channels, impedance channels, and thelike. The antibodies for the various dedicated lineages may beilluminated by different fluorochromes. Fluorochromes that may find usein a multicolor analysis include phycobiliproteins, e.g., phycoerythrinand allophycocyanins; fluorescein; and Texas red. The cells may also beselected against dead cells, by employing dyes that selectivelyaccumulate in dead cells (e.g., propidium iodide and 7-aminoactinomycinD (7-AAD)). Preferably, the cells are collected in a medium comprisingabout 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA). See,for example, Fallon et al. (2003) Br. J. Haematol. 121:1, hereinincorporated by reference.

Other techniques for positive selection may be employed, which permitaccurate separation, such as affinity columns, and the like. The methodof choice should permit the removal of the non-progenitor cells to aresidual amount of less than about 20%, less than about 15%, less thanabout 10%, or less than about 5% of the desired population of stemcells.

The stem cell populations of the invention can be isolated from stemcell sources using a variety of methods, including those describedherein and exemplified below. For example, in one embodiment, a two-stepprocedure can be used. For example in a first step, stem cells arepositively selected by first sorting for cellular expression ofALDH^(br), for example by using FACS. In a second step, cells are eitherpositively selected by sorted for expression of cell surface markerssuch as CD105, HLA-DR, CD41, or negatively sorting for lack ofexpression of cell surface markers such as lineage commitment markers.

Alternatively, a three-step isolation procedure can be used. In a firststep, mature mononuclear cells, for example, which express any of thedifferent surface antigens associated with specific lineage commitment,can be eliminated using a commercially available kit from StemCellTechnologies. The resulting preparation, designated Lin⁻, is essentiallydevoid of cells expressing any of the following surface antigens:glycophorin A (glyA), CD2, CD3, CD14, CD16, CD19, CD24, CD56, and CD66b.In a second step, a subset of Lin⁻ cells can be sorted by flow cytometryafter staining with, for example, BODIPY aminoacetaldehyde, to obtainALDH^(br) Lin⁻ and ALDH^(lo)Lin⁻. In a third step, CD34⁺ cells can besorted from the ALDH^(br) Lin⁻ fraction using, for example, flowcytometry, to yield ALDH^(br) CD34⁻ Lin⁻ cells and ALDH^(br) CD34⁺Lin⁻cells. This preparation can further be sorted for cells positivelyexpressing CD13, CD33, CD35, CD38, CD41, CD45, CD90, CD105, CD117,CD133, CD135, CD138, HLA-DR, or any combinations thereof.

In an alternative embodiment, the isolation procedure involves the useof a single negative depletion step. In accordance with this procedure,cells expressing Lin lineage markers are eliminated in a single stepusing appropriate antibodies.

Regardless of the isolation procedure used, the resulting cells haveproperties of multilineage stem cells. By “multilineage stem cells” isintended cells capable of multilineage development into cells such asmesenchymal stem cells (MSC), hematopoietic stem cells (HSC), orself-renewing progenitor cells that are themselves capable of MSC or HSCdevelopment. By “multilineage development” is intended capable ofdeveloping into any differentiated tissue including, but not limited to,blood cells (including lymphocytes, myelocytes, erythrocytes, andplatelets); bone (including osteocytes, osteoblasts, and osteoclasts);marrow stroma; cartilage (including chrondrocytes, chrondroblasts, andchrondroclasts); cardiac, smooth, and skeletal muscle (includingmyocytes and cardiomyocytes); tendon; nerves (including oligodendrocytesand neurons); vascular tissue (including angiocytes and theendothelium); fat (including adipocytes); fibroblasts; liver cells; gutand lung epithelial cells; cornea (including corneocytes; and dermis(including dermal cells). By “hematopoietic stem cells” is intended stemcells that differentiate into blood cells. By “mesenchymal stem cells”is intended stem cells that differentiate into non-blood cells. SPC andASPC comprise multilineage stem cells capable of developing into eithermesenchymal stem cells or hematopoietic stem cells.

Hematopoietic stem cells do not express cell surface markers forimmature and mature lymphocytes, erythrocytes, or myeloid cells, unlikelineage-committed precursors. More specifically, they have no detectableexpression of surface markers of myeloid cells (CD33), B-lymphocytes(CD19, HLA-DR), T-lymphocytes (CD3, CD4, CD8), NK cells (CD16, CD56), orerythroid cells (CD71), where expression is detected usingfluorescence-conjugated antibodies. Hematopoietic stem cells also do nothave detectable expression of the early lymphoid markers CD1a, CD2, CD5,or CD10, which are present on committed lymphoid precursors.Advantageously, the primitive hematopoietic stem cells of the inventiondo not have detectable expression of any of the following antigens: CD3,CD4, CD8, CD13, CD14, CD16, CD19, CD25, CD33, CD56, CD71, CD124, CD130,CD138, or glyA. Preferably, the ALDH^(br) stem cells are also capable ofsustaining long-term hematopoiesis in mice (see, for example, Goodell etal. (1996) J. Exp. Med. 183:1797). More preferably, they are in G₀, acell cycle state that is believed to be a property of primitivehematopoietic cells (Spangrade et al. (1990) Proc. Natl. Acad. Sci. USA87:7433).

In one embodiment, the stem cells of the invention comprise ALDH^(br)cells derived from bone marrow. These ALDH^(br) cells can be used togenerate any cell of the hematopoietic lineage, including, but notlimited to, myeloid cells (such as platelets, megakaryocytes, and redbloods cells) and lymphoid cells (such as T cells, B cells, NK cells,and antigen presenting cells). In an alternative embodiment, the stemcell of the invention can comprise ALDH^(br) CD105⁺ cells isolated fromany stem cell source, not just bone marrow. These cells are also capableof generating any cell of the hematopoietic lineage, including, but notlimited to, myeloid cells (such as platelets, megakaryocytes, and redbloods cells) and lymphoid cells (such as T cells, B cells, NK cells,and antigen presenting cells). In an alternative embodiment, theALDH^(br) cells derived from bone marrow or the ALDH^(br)CD 105⁺ cellsisolated from any stem cell source comprise cells with a low granularityas measured in the side scatter channel of a flow cytometer (SSC^(lo)).

Mesenchymal stem cells (MSC) have been characterized using panels ofantibodies much like hematopoietic stem cells. MSC generally lackexpression of CD14, CD34, and CD45. MSC are generally positive for CD105and CD73. Other markers used by researchers to identify culturedmesenchymal cells include positive expression of such markers as CD29,Thy-1 (CD90), CD115, CD144, CD166, and HLA-A, B, or C. Functionally, MSCcan be tested in vitro for their ability to differentiate intoadipogenic, osteogenic, myogenic, and chondrogenic cell colonies.

As disclosed supra, the stem cells of the invention comprise ALDH^(br)cells derived from bone marrow. These ALDH^(br) cells can be also usedto at least generate any cell of the mesenchymal lineage, including, butnot limited to, bone (including osteocytes, osteoblasts, andosteoclasts), marrow stroma, cartilage (including chrondrocytes,chrondroblasts, and chrondroclasts), cardiac, smooth, and skeletalmuscle (including myocytes and cardiomyocytes), tendon, nerves(including oligodendrocytes and neurons), vascular tissue (including theendothelium), fat (including adipocytes), fibroblasts, and dermis. In analternative embodiment, the stem cell of the invention can compriseALDH^(br) CD105⁺ cells isolated from any stem cell source, not just bonemarrow. These cells are also capable of generating any cell of themesenchymal lineage, including, but not limited to, bone (includingosteocytes, osteoblasts and osteoclasts), marrow stroma, cartilage(including chrondrocytes, chrondroblasts, and chrondroclasts), cardiac,smooth, and skeletal muscle (including myocytes and cardiomyocytes),tendon, nerves (including oligodendrocytes and neurons), vascular tissue(including the endothelium), fat (including adipocytes), fibroblasts,and dermis. In an alternative embodiment the ALDH^(br) cells derivedfrom bone marrow or the ALDH r CD105⁺ cells isolated from any stem cellsource comprise cells with a low granularity as measured in the sidescatter channel of a flow cytometer (SSC^(lo)).

In some embodiments, the stem cell populations of the invention isolatedfrom bone marrow, for example, are ALDH_(dim) and comprise cells thatare CD45 negative. In other embodiments, the stem cell populations ofthe invention isolated from bone marrow, for example, are ALDH^(br) andcomprise cells that are CD45 negative. In yet other embodiments, thestem cell population isolated from umbilical cord blood and mobilizedperipheral blood, for example, are ALDH^(br) and are CD45 negative. Inyet other embodiments, the stem cell population isolated from umbilicalcord blood and mobilized peripheral blood, for example, are ALDH^(dim)and are CD45 negative.

Functional assays for stem cells include both in vitro and in vivomethods. For in vitro tests, cultures are seeded with cells from tissuesunder conditions that favor the differentiation of SPC or ASPC intospecific types of tissue cells. See, for example, Eaves, “Assays ofHematopoictic Progenitor Cells” in Williams (1995) Hematology at L22-6(5^(th) ed., E. Beutler et al. eds.); Petzer et al. (1996) Proc. Natl.Acad. Sci. USA 93:1470; Mayani et al. (1993) Blood 81:3252; Hogge et al.(1997) Br. J. Haematol. 96:790; Lazarus et al. (1995) Bone MarrowTransplant. 16:557; Pittenger et al. (1999) Science 284:143.

Usually, ASPC will multiply a few times in culture as they differentiateto form a colony of differentiated cells. In some assays, such as assaysinvolving blood cell differentiation, the developmental potential of acell that initiated the colony is inferred from the range of phenotypeswithin the colony. Alternatively, cell cultures may be sampled andsubcultured into a different culture system or into animal models, suchas immunodeficient NOD-SCID mice or sheep embryos, to determine ifadditional phenotypes can be induced from APSC that remain in theindividual colony. See, for example, Almeida-Porada et al. (2000) Blood95:3620; Lewis et al. (2001) Blood 97:3441; Rice et al. (2000)Transplantation 69:927; Ishikawa et al. (2002) Exp. Hematol 30:488. ASPCthat give rise to multiple phenotypes are usually considered moreprimitive, multipotent progenitors than cells that give rise to fewerphenotypes.

Based on these assays, the choice of a starting stem cell source coupledwith the specific lineage-preferred cell population allows for a varietyof therapeutic applications using one or a few ASPC types. For example,ALDH^(br) cell populations that have been isolated by sorting BM includemore mesenchymal stem cells capable of giving rise to non-hematopoietictissues than do ALDH^(br) cell populations derived from MPB or UCB basedon the increased percentage of cells expressing early mesenchymal stemcell markers such as CD105. See, Example 1, Table 1, herein below.

The stem cell populations of the present invention have application in avariety of therapies and diagnostic regimens. They are preferablydiluted in a suitable carrier such as buffered saline before injection.The cells may be administered in any physiologically acceptable vehicle,normally intravascularly, although they may also be introduced into boneor other convenient site where the cells may find an appropriate sitefor regeneration and differentiation (e.g., thymus). Usually, at least1×10⁵ cells/kg and preferably 1×10⁶ cells/kg or more will beadministered. See, for example, Sezer et al. (2000) J. Clin. Oncol.18:3319 and Siena et al. (2000) J. Clin. Oncol. 18:1360. The cells maybe introduced by injection, catheter, or the like. If desired,additional drugs such as 5-fluorouracil and/or growth factors may alsobe co-introduced. Suitable growth factors include, but are not limitedto, cytokines such as IL-2, IL-3, IL-6, IL-11, G-CSF, M-CSF, GM-CSF,gamma-interferon, and erythropoietin. In some embodiments, the cellpopulations of the invention can be administered in combination withother cell populations that support or enhance engraftment, by any meansincluding but not limited to secretion of beneficial cytokines and/orpresentation of cell surface proteins that are capable of deliveringsignals that induce stem cell growth, homing, or differentiation. Inthese embodiments, less than 100% of the graft population comprise theALDH^(br) stem cells.

As the isolated cells of the invention are capable of engraftinghematopoietic stem cells, they are suitable for both transplantation andgene therapy purposes. Since the populations of cells can be sorted intoboth lineage-committed and lineage-uncommitted cells, each populationwill have different, but non-mutually exclusive, therapeuticapplications. In some embodiments, lineage-committed cells will beuseful for reconstitution or augmentation of lymphoid and/or myeloidcell populations. For example, indications to be treated includeimmunodeficiencies and stem cell transplantations. The progenitor cellsare particularly useful during stem cell transplantation to decrease thelag time between the transplantation and repopulation of thehematopoietic cells. Methods of obtaining the progenitor cells aredescribed herein. Methods of administering stem cells to patients arewithin the skill of one in the art as noted herein above.

In some embodiments, identification of a lymphoid-restricted progenitorcell population allows assessment of this population as to the origin ofdisease, e.g., leukemia, and therefore is useful in purging such cellsfrom an autologous graft. In other embodiments, lineage-committedprecursor cells may be useful as a cellular graft to increase thepatient's ability to mount an immune response. In some instances, it maybe advantageous to use precursors in a vaccine strategy whereby thecells are loaded with antigen and injected to induce a specific immuneresponse. Alternatively, the injected cells may be used to inducetolerance to a specific exogenously introduced antigen.

Lineage-committed precursors may be used for a variety of gene therapyapproaches where expression of the exogenous genetic capability isdesired in lymphoid and/or myeloid lineages. Lineage-committedprogenitor cells will also be preferred in cases where it is desired tohave temporary rather than permanent expression of the exogenous geneticcapability. Also, in some instances, gene transfer is likely to be moreefficient in lineage-committed progenitors because progenitor cellscycle more actively than stem cells and retroviral vectors are known torequire actively cycling cells for efficient integration of therecombinant DNA.

In addition, it would be advantageous to use lymphoid progenitorscompared to using mature lymphoid cells for gene therapy. Currently, Tcell gene therapy requires ex vivo expansion of T cells with cytokines.Upon reinfusion, the modified T cells often do not home properly totheir target organs and may become trapped in (and cleared by) thelungs, liver, or spleen. This improper homing may be due to alterationof the membranes during the ex vivo processing, downregulation of homingreceptors, or the like. Use of modified progenitor cells would obviatethe necessity of ex vivo expansion of the effector T cells, and thusobviate concerns of altered trafficking and persistence in vivo. Inaddition, the use of modified progenitors will allow amplification inprogeny cell numbers, thereby reducing the need for ex vivo expansionand reducing the frequency of administration.

In other embodiments, it is preferable to use non-lineage-committedcells or more primitive stem cells. Non-lineage committed cells areuseful for treating diseases (and related therapeutic sequelae) causedby the impaired function of bone marrow cells. Examples of such diseasesinclude, but are not limited to, lymphoma, multiple myeloma, breastcancer, testicular cancer, leukemias, congenial hemolytic anemias (e.g.,thalassemia), and some immunodeficiency diseases, e.g., acute leukemia,Hodgkin's and non-Hodgkin's lymphoma, and neuroblastoma. It has alsobeen shown that the hematologic toxicity sequelae observed with multiplecycles of high-dose chemotherapy is relieved by conjunctiveadministration of autologous hematopoietic stem cells. Diseases forwhich reinfusion of stem cells has been described include acuteleukemia, Hodgkin's and non-Hodgkin's lymphoma, neuroblastoma,testicular cancer, breast cancer, multiple myeloma, thalassemia, andsickle cell anemia (Cheson et al. (1989) Ann. Intern. Med. 30 110:51;Wheeler et al. (1990) J. Clin. Oncol. 8:648; Takvorian et al. (1987) N.Engl. J. Med. 316:1499; Yeager, et al. (1986) N. Eng. J. Med. 315:141;Biron et al. (1985) In Autologous Bone Marrow TransplantationProceedings of the First International Symposium, Dicke et al., eds., p.203; Peters (1985) ABMT, id. at p. 189; Barlogie, (1993) Leukemia7:1095; Sullivan, (1993) Leukemia 7:1098-1099).

For example, in cancer patients, sorting stem cells of the inventionsuch as ALDH^(br) bone marrow derived cells or ALDH^(br) CD105⁺ cellsfrom any source separates stem cells from cancer cells prior toreintroduction into the patient. In these patients undergoing autologoustransplantation, such separation can be used to reduce the chance thatcancer cells are returned to the patient (Jones et al. (1987) Blood70:1490; Colvin In: Hematopoietic Cell Transplantation 217 (Forman ed.,1999), and Russo and Hilton (1989) in Enyzmology and Molecular Biologyof Carbonyl Metabolism at 65 (Weiner and Flynn, eds.). In anotherembodiment, purified autologous ALDH^(br) stem cells of the inventioncan be ex vivo expanded prior to reintroduction into the patient tohasten lymphoid, erythroid, and platelet engraftment. Ex vivo expansioncan be effected by growth in defined cytokines, on stromal layers,and/or in bioreactors (Emerson et al. (1996) Blood 87:3082). Inaddition, the incidence of graft failure can be reduced. This isbeneficial for cancer patients undergoing autologous transplantation,for gene therapy, and for patients suffering from auto-immune disorders.

In some embodiments, while not being bound to any mechanism of action ortheory, the population of non-lineage committed stem cells of theinvention may be used to hasten healing of injuries, repair injuries,and regenerate tissues. As the cell populations of the invention candifferentiate into mesenchymal tissue, the stem cells of the inventioncan be used beneficially in a wide variety of diseases not associatedwith hematopoiesis, for example joint regeneration for arthritis; boneregeneration for osteoporosis, osteoimperfecta, and periodontitis;angiogenesis and endothelial cell regeneration for ischemic injury;muscle re-growth for degenerative muscle disease such as musculardystrophy and cardiac ischemia; neural cell regrowth in spinal cord andbrain injury; regeneration of oligodendrocytes in multiple sclerosis; aswell as surgeries where tissue regrowth is beneficial such as plasticsurgery, orthopedic surgery, and surgical tissue removal. Thus, themesenchymal stem cells of the invention can used to produce, in someembodiments, new endothelium, new cardiomyocytes, new neurons andoligodendrocytes, and new bone and cartilage. For example, it has beenshown that mesenchymal stem cells grow endothelium, and that mesenchymalgrafts treat restenosis and ischemic heart injury. See, for example,Hristov et al. (2003) Trends Cardiovasc Med. 13:201; Sata (2003) Trendsin Cardiovasc. Med. 13:249; Isner et al. (2001) Ann. NY Acad. Sci.953:75; Abbott and Giordano (2003) J. Nucl. Cardiol. 10:403; Forresteret al. (2003) Circulation 108:1139. It has also been shown thatmesenchymal stem cell grafts promote regrowth of damaged neurons afterneurological damage or disease. See, for example, Gage (2000) Science287:1433; Shin et al. (2001) Blood 98:2412; Zhao et al. (2003) BrainRes. Protocols 11:38. It has also been shown that mesenchymal stem cellgrafts promote new bone and cartilage growth. See, for example,Pittenger et al. (1999) Science 284:143, and U.S. Pat. No. 6,541,024.

In some embodiments, the isolated stem cell populations of the inventionare suitable for use in gene therapy. For example, following isolationof autologous ALDH^(br) cells from bone marrow or ALDH^(br)CD105⁺ fromany stem cell source, the isolated cells are exposed to a gene deliveryvector, and the genetically modified stem cells are reinfused into thepatient. Gene therapy protocols using genetically modified stem cellsare known in the art. See, for example, Smith (1992) Hematother. 1:155.This approach can involve ex vivo culture or the use of vectors capableof transferring genes into non-dividing cells, thereby rendering ex vivoculture unnecessary. By the term “ex vivo gene therapy” is meant the invitro transfection or retroviral infection of stem cells prior tointroducing the transfected stem cells into a mammal. Gene therapy canbe useful in treating, for example, congenital diseases, which include,but are not limited to, adenosine deaminase deficiency, recombinasedeficiency, recombinase regulatory gene deficiency, and sickle cellanemia. In treating sickle cell anemia, for example, the mutant β-globingene is replaced or supplemented with either the wild-type globin geneor an anti-sickling globin gene. In the treatment of cancer, drugresistance genes can be introduced into cells to confer resistance tocytotoxic drugs. This can reduce the incidence and severity ofmyelosuppression. For the treatment of infectious diseases, includingHIV, anti-viral genes can be introduced into stem cell populations ofthe invention so that they are rendered resistant to the virus (see, forexample, Gilboa and Smith (1994) Trends in Genetics 10:139).

In some embodiments, isolation of ALDH^(br) cells from bone marrow orALDH^(br)CD 105⁺ from any stem cell source results in the elimination ofpre-T-cells that cause graft versus host disease (GvHD). Thiselimination from the stem cell population of the invention can beexpected to reduce the incidence and severity of GvHD in recipients ofallogeneic transplants. See, for example, Ho and Soiffer (2001) Blood98:3192.

Isolated ASPC can be ex vivo expanded to hasten neutrophil, erythroid,and platelet engraftment after allogeneic transplantation. In addition,the incidence of graft failure can be reduced. This is likely to beparticularly important for recipients of umbilical cord bloodtransplants, where small cell doses limit the success oftransplantation. Techniques for ex vivo expansion are well described inthe art. See, for example, McNiece and Briddle (2001) Exp. Hematol.29:3; McNiece et al. (2000) Exp. Hematol. 28:1186; Jaroscak et al.(2003) Blood 101:5061.

In some embodiments, successful engraftment with ALDH^(br) cells frombone marrow or ALDH^(br)CD105⁺ from any stem cell source can also beexpected to induce tolerance to alloantigens. Prior induction oftolerance enhances the chance of permanent engraftment of subsequentsolid organ transplants that originate from the same donor.

It will be appreciated that the stem cell populations of the presentinvention can be used as sources of new genes (e.g., for cytokines andcytokine receptors), including genes important in growth anddevelopment.

In addition to their application in treatment and diagnosis strategies,the stem cell populations of the present invention can be used inscreening protocols to identify agents that can be used, for example, topromote differentiation or growth and/or engraftment of stem cells. Inone such protocol, ALDH^(br) cells from bone marrow or ALDH^(br)CD105⁺from any stem cell source are contacted with a test compound suspectedof inducing differentiation, and the ability of the test compound toeffect differentiation is determined (using, for example, microscopicand flow cytometric examination). In another screening protocol,ALDH_(br) cells from bone marrow or ALDH^(br)CD105⁺ from any stem cellsource are contacted with a test compound suspected of inducingproliferation and/or engraftment and the ability of the test compound toeffect proliferation and/or engraftment is determined, for example,using in vitro long-term colony assays or in vivo immunodeficient micemodels (e.g., SCID/NOD mice). See, for example, Peault et al. (1993)Leukemia 7:s98-101.

The invention also relates to kits that can be used to prepare the stemcell populations of the present invention, where, in some embodiments,the populations comprise ALDH^(br) cells from bone marrow orALDH^(br)CD105⁺ from any stem cell source. The kits can comprise an ALDHsubstrate disposed in a single container and antibodies that can be usedto effect direct isolation of the cells in separate containers. In apreferred embodiment, the kit includes at least antibodies specific forlineage-specific markers disposed within one or more container means.These antibodies can be used in conjunction with those present in theStemCell Technologies kit to achieve, for example, about 80%purification or higher. Advantageously, the kit also includes antibodiesspecific for HLA-DR. Any or all of antibodies specific for CD2, CD3,CD8, CD10, CD13, CD14, CD16, CD19, CD33, CD34, CD35, CD38, CD41, CD45,CD56, CD71, CD105, CD117, CD124, CD127, CD130, CD138, and glyA can alsobe included, disposed within at least one container means. The kit canalso include, disposed within a container means, anti-CD7 antibodies.

The antibodies of the kits are disposed within a container means and thekit can further include ancillary reagents (e.g., buffers and the like)suitable for carrying out the isolation protocols.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Tissue Processing and Phenotyping

Directly conjugated fluorescent antibodies were employed for analyses ofcell surface antigens. Those used included antibodies directed againstCD3 (Leu4; Becton Dickinson clone SK7); CD7 (Leu9; Becton Dickinsonclone M-T701); CD10 (Beckman Coulter/Immunotech clone ALB1); CD13(Beckman Coulter/Immunotech clone SJ1D1); CD14 (BeckmanCoulter/Immunotech clone RM052); CD19 (Leu12; Pharmingen clone WM15);CD33 (LeuM9; Becton Dickinson clone M-φP9); CD34 (Becton Dickinson clone8G12); CD35 (Pharmingen clone E11); CD38 (Leu17; Becton Dickinson cloneHB7); CD41 (Caltag clone VIPL3); CD45 (Miltenyi clone 5B1); CD56 (Leu19;Becton Dickinson clone My31); CD90 (Pharmingen; clone 5E10); CD105(Caltag clone SN6); CD117 (Becton Dickinson clone 104D2); CD127 (BeckmanCoulter/Immunotech clone R34.34); CD133 (Miltenyi clone AC 133); CD135(Pharmingen clone 4G8); CD 138 (IQ products clone B-B4); HLA-DR (BectonDickinson clone L243); and glyA (Becton Dickinson clone GAR-2).

Fresh bone marrow samples from ten normal donors (Cambrex) were dilutedin ammonium chloride 1:40 by volume (0.17 M NH₄Cl containing 10 mMTris-HCl, pH 7.2 and 200 mM EDTA final concentration) and incubated at4° C. for 30 minutes to lyse erythrocytes. Cells were pelleted bycentrifugation, resuspended at 1×10⁷/ml, and reacted for aldehydedehydrogenase activity using the ALDEFLUOR™ kit (StemCo Biomedical,Inc.). Cells were suspended in assay buffer, and 50 ml of substrate/mlwere added to the cells and mixed immediately. 100 ml of cells wereremoved from the substrate tube and added to thediethylaminobenzaldehyde (DEAB) control tube. Cells were incubated at37° C. for 30 minutes. 3-4×10⁶ cells were placed in each immunophenotypetube and incubated for an additional 15 minutes at 4° C. with theappropriate monoclonal antibodies. After incubation, 1 ml of assaybuffer was added to each tube to wash the cells, and the cells werepelleted by centrifugation. The supernatant was removed, and the cellswere resuspended in 300-500 ml of assay buffer.

Fresh UBC samples were red blood cell depleted using hetastarch andammonium chloride lysis or hetastarch and ficoll. 20 ml of cord bloodwas diluted with an equal volume of PBS and 8 mls of hetastarch (HetaSep; Stem Cell Technologies, Inc.). Samples were mixed and incubated for60 minutes at room temperature. The white blood cell fraction wasremoved from each tube and pelleted by centrifugation. Recovered cellswere resuspended in 30 ml of ammonium chloride and incubated at 37° C.for 15-30 minutes or passed over a ficoll gradient to removecontaminating red blood cells. Cells were washed, diluted, andphenotyped with monoclonal antibodies and ALDEFLUOR™ as described above.

Fresh mobilized peripheral blood samples from normal donors and alsofrom cancer patients (i.e., post G-CSF infusion) were diluted 1:20 inammonium chloride and incubated for 15-30 minutes at 37° C. See, Fieldset al. (1994) Cancer Control 1:213 and Elfenbein et al. (1995) Annal. NYAcad. Sci. 770:315 for patient procedures. The recovered cells were thenwashed, diluted, and phenotyped with monoclonal antibodies andALDEFLUOR™ as described above.

All human samples were obtained using technical and informed consentprocedures that complied with relevant regulations and were approved bylocal institutional review boards.

Multiparameter phenotyping data was acquired on a Becton DickinsonFACSCalibur using an argon laser emission at 488 nm to excite theALDEFLUOR™ reaction product (fluorescence detected in FL1 channel),phycoerythrin-conjugated antibodies to a large panel of markers (seeTable 1; fluorescence detected in FL2 channel); 7-aminoactinomycin D wasused to stain and discriminate dead cells (fluorescence detected in FL3channel), and a HeNe laser was used to exciteallophycocyanine-conjugated CD34 (fluorescence detected in FL4).Fluorescence was carefully compensated in multiparameter analysis tocorrect for spillover of signals among channels (see Table 1 forcompensation controls). The ALDH^(br)SSC^(lo) cell population wasdefined by gating on forward and side scatter and FL1. A large data filewas acquired such that >2000 events were contained in theALDH^(br)SSC^(lo) gate.

Table 1 shows the percentage of the cells within the ALDH^(br)SSC^(lo)window that express the given marker and also the percentage of thetotal cells in the sample that express the marker that fall outside theALDH^(br) window. Thus, CD7, primarily a T cell marker, and CD45, apan-blood cell marker, fall primarily outside the window, while thepercentage of stem cell markers such as CD34 and CD133 are increasedwithin the window relative to ungated cells. Notably, not all of thecells expressing stem cell markers are in the window. Therefore, ALDHdefines subsets of these stem cell markers, and the populations aredifferent in the three sources. The distribution of the markers in theALDH^(br) window varies both among tissues and among samples of a giventissue (especially for cord blood), which suggests that the ALDH markergives more information about the physiological and functional state ofthe stem cells.

Table 2 shows the percentage of CD34⁺ cells that also express each ofthe other markers shown in each tissue. In this table, all markers wereanalyzed with regard to CD34 and ALDH expression.

Table 3 shows the overall expression of the markers in the threetissues.

Table 4A-C summarizes the expression of CD markers in different gatesfor bone marrow, umbilical cord blood, and peripheral blood,respectively. The overall expression in the tissue, the expression inthe ALDH^(br) gate, and the expression in the CD34⁺ gate are shown.

Within this gate (ALDH^(br)SSC^(lo)), it was observed that BM includesmore cells capable of giving rise to non-hematopoietic tissues (e.g.,positive for CD34, CD105, CD117, and CD133) than populations derivedfrom MPB or UCB based on the phenotypic profile. Moreover, CD105 isexpressed to a greater extent in the bone marrow ALDH^(br) cellpopulation than in other grafts. The CD105⁺ and CD133⁺ cells are verybright for ALDH in the bone marrow, even brighter than CD34 positivecells. It is possible that CD133⁺ cells are CD105 positive, but that notall CD105⁺ cells are CD133 positive. Also, the SSC^(lo) window does notcapture all of the CD105/CD133 ALDH cells in marrow, although it doescapture the CD34⁺ cells, which is different in UCB and PBSC. Thescatterplots and comparative data supporting these conclusions are shownin FIGS. 1-6.

TABLE 1 Percentage of Cells Expressing CD Marker Shown Mean and StandardDeviation Summary Bone Marrow (n = 10) Cord Blood (n = 7) PeripheralBlood (n = 16) % of cells % of cells % of cells expressing marker % ofALDHbr expressing marker % of ALDHbr expressing marker % ofALDH^(br cells) that are NOT cells expressing that are NOT cellsexpressing that are NOT expressing marker ALDH^(br) marker ALDH^(br)marker ALDH^(br) CD Marker Mean S.D. Mean S.D. Mean S.D. Mean S.D. MeanS.D. Mean S.D. CD3 1.36 0.788 99.91 0.098 1.95 1.249 99.96 0.043 1.471.426 99.91 0.115 CD7 1.31 0.798 99.78 0.384 7.36 14.051 99.92 0.0831.02 0.845 99.84 0.236 CD10 1.29 1.234 98.35 4.383 0.46 0.410 98.392.425 0.47 0.721 94.22 14.119 CD13 7.96 8.649 99.59 0.401 16.33 21.49599.21 1.537 24.84 26.139 98.67 2.962 CD14 1.68 1.755 99.78 0.202 1.131.359 99.97 0.021 0.66 0.798 99.96 0.051 CD19 1.53 1.697 99.07 1.9340.55 0.660 99.76 0.555 0.60 0.762 98.53 3.092 CD33 7.55 7.510 98.573.470 12.33 13.595 99.39 1.091 18.71 22.23 99.22 1.134 CD34 68.96 14.63054.19 12.978 81.41 5.252 25.60 11.454 94.85 4.405 24.39 25.683 CD35 1.571.239 99.43 1.500 1.01 0.974 99.95 0.053 2.37 2.65 99.73 0.667 CD3868.75 16.872 96.09 1.754 49.20 21.900 99.31 0.793 69.17 26.779 97.762.452 CD41-C 27.22 18.522 98.95 0.695 16.89 19.567 99.43 0.521 27.2623.166 98.52 2.910 CD45-M 54.62 18.533 99.26 0.395 78.28 20.454 99.160.726 86.08 21.323 98.56 2.128 CD56-BD 2.16 1.398 97.39 2.762 4.64 9.89999.40 0.936 1.62 2.269 98.55 3.868 CD90 3.22 3.532 93.87 6.810 8.0014.188 98.88 0.984 2.52 2.356 89.10 15.818 CD105 10.28 11.173 75.1822.390 0.82 0.686 94.87 10.439 1.03 1.188 93.06 16.374 CD117 43.7823.037 72.54 14.873 45.96 26.423 43.51 8.297 18.99 18.045 45.61 17.208CD127 0.74 0.688 76.77 19.260 0.39 0.255 88.66 14.759 0.54 0.657 73.8927.193 CD133 4.06 4.626 61.29 32.003 29.61 21.858 37.21 14.145 36.8026.149 26.23 16.888 CD135 0.84 0.704 84.93 18.976 1.09 1.428 90.9315.926 0.58 0.717 81.56 19.431 CD138* 0.71 0.740 88.36 17.536 0.11 99.670.57 0.582 76.98 24.293 HLA-DR 38.43 16.437 97.46 1.620 65.79 20.13698.02 1.952 64.89 26.688 95.61 4.872 GLY-A 5.67 4.942 98.02 1.942 1.460.479 98.47 1.994 0.69 0.694 96.06 6.321 ALDH 0.97 0.598 0.80 0.556 1.111.084 *For Bone Marrow CD138: n = 7 *For Cord Blood CD138: n = 1 *ForPeripheral Blood CD138: n = 10

TABLE 2 Percentage of CD34+ Cells Expressing CD Marker Shown Bone Marrow(n = 10) Cord Blood (n = 7) Peripheral Blood (n = 16) CD Marker MeanSTDEV Mean STDEV Mean STDEV CD3 0.69 0.348 3.15 1.863 1.86 2.371 CD70.78 0.444 6.30 6.090 1.29 0.976 CD10 8.49 8.179 1.07 1.334 0.61 0.737CD13 6.09 7.420 14.60 21.476 25.16 25.962 CD14 0.26 0.317 0.25 0.1880.57 0.556 CD19 3.17 3.328 1.53 0.872 0.67 0.837 CD33 6.52 4.710 9.7012.607 18.25 22.459 ALDH 47.19 11.882 74.40 11.454 83.41 11.946 CD350.48 0.424 1.22 1.078 2.06 2.040 CD38 69.92 20.245 47.80 22.845 67.6527.161 CD41-C 25.35 19.138 16.51 21.023 26.94 23.251 CD45-M 51.03 22.60475.61 25.070 84.27 23.342 CD56-BD 0.79 0.658 3.17 7.043 1.51 2.186 CD901.75 3.218 6.14 11.781 1.75 1.868 CD105 1.34 1.256 0.23 0.187 0.78 1.128CD117 32.91 14.833 45.36 25.923 17.93 18.403 CD127 0.27 0.181 0.14 0.1590.40 0.489 CD133 2.78 4.016 30.74 24.161 35.63 26.734 CD135 0.16 0.0940.34 0.401 0.43 0.632 CD138 0.13 0.151 0.09 n/a 0.43 0.584 HLA-DR 49.7618.578 64.26 22.024 62.44 26.427 GLY-A 0.38 0.228 0.85 0.556 0.51 0.611ALDH 0.97 0.599 0.80 0.556 1.11 1.085 R3 iso 0.45 0.304 0.47 0.146 0.300.325 R5 iso 0.19 0.288 0.78 0.780 0.19 0.261 R2 pe iso 3.59 8.143 0.930.510 0.79 0.815 G7 pe iso 13.51 21.887 3.37 3.064 4.57 6.799

TABLE 3 Percentage of Ungated Cells Expressing Shown Markers Bone MarrowCord Blood (n = 10) (n = 6) Peripheral Blood CD Marker Mean STDEV MeanSTDEV Mean STDEV (n = 16) CD3 14.72 3.727 43.69 14.643 21.81 18.080 CD78.38 2.719 43.76 17.460 14.85 15.860 CD10 3.73 2.290 0.79 0.847 0.720.933 CD13 19.42 8.313 22.12 10.275 45.08 27.214 CD14 5.24 1.362 22.623.440 25.43 11.399 CD19 2.68 1.751 6.45 3.990 4.69 8.967 CD33 15.519.257 23.73 3.985 25.98 17.523 CD34 1.40 0.500 0.90 0.517 0.98 1.082CD35 9.58 8.285 20.08 8.910 41.67 21.568 CD38 17.66 3.871 71.88 18.73536.31 18.805 CD41-C 26.32 14.980 22.33 5.725 41.81 19.781 CD45-M 70.5113.339 88.84 12.651 96.93 4.692 CD56-BD 1.07 0.854 5.90 7.961 3.98 4.527CD90 1.88 3.681 6.24 10.195 0.72 0.347 CD105 0.53 0.367 1.44 2.131 2.364.736 CD117 1.52 0.582 0.67 0.500 0.24 0.308 CD127 0.07 0.053 0.59 1.0121.03 2.686 CD133 0.12 0.113 0.32 0.255 0.27 0.151 CD135 0.15 0.119 0.340.463 0.10 0.080 CD138 0.07 0.060 0.37 #DIV/0! 0.04 0.012 HLA-DR 14.922.690 31.27 6.338 21.49 10.799 GLY-A 9.16 5.722 3.07 2.110 0.47 0.379ALDH 0.97 0.599 0.80 0.556 1.11 1.085 n = 9 R3 iso 0.45 0.304 0.47 0.1460.30 0.325 R5 iso 0.19 0.288 0.78 0.780 0.19 0.261 R2 pe iso 3.59 8.1430.93 0.510 0.79 0.815 G7 pe iso 13.51 21.887 3.37 3.064 4.57 6.799 CD45APC 79.57 11.861 89.39 10.992 96.96 2.178 AB45 APC 71.45 25.804 66.3324.573 79.89 21.902

TABLE 4A Summary of Bone Marrow (All Gates) Bone Marrow ALDH CD+ CD34CD+CD+ CD Marker Mean % St. Dev. Mean % St. Dev. Mean % St. Dev. CD3 1.360.788 0.69 0.348 14.72 3.727 CD7 1.31 0.798 0.78 0.444 8.38 2.719 CD101.29 1.234 8.49 8.179 3.73 2.290 CD13 7.96 8.649 6.09 7.420 19.42 8.313CD14 1.68 1.755 0.26 0.317 5.24 1.362 CD19 1.53 1.697 3.17 3.328 2.681.751 CD33 7.55 7.510 6.52 4.710 15.51 9.257 ALDH 68.96 14.630 47.1911.882 1.40 0.500 CD35 1.57 1.239 0.48 0.424 9.58 8.285 CD38 68.7516.872 69.92 20.245 17.66 3.871 CD41-C 27.22 18.522 25.35 19.138 26.3214.980 CD45-M 54.62 18.533 51.03 22.604 70.51 13.339 CD56-BD 2.16 1.3980.79 0.658 1.07 0.854 CD90 3.22 3.532 1.75 3.218 1.88 3.681 CD105 10.2811.173 1.34 1.256 0.53 0.367 CD117 43.78 23.037 32.91 14.833 1.52 0.582CD127 0.74 0.688 0.27 0.181 0.07 0.053 CD133 4.06 4.626 2.78 4.016 0.120.113 CD135 0.84 0.704 0.16 0.094 0.15 0.119 CD138 0.71 0.740 0.13 0.1510.07 0.060 HLA-DR 38.43 16.437 49.76 18.578 14.92 2.690 GLY-A 5.67 4.9420.38 0.228 9.16 5.722

TABLE 4B Summary of Cord Blood (All Gates) Cord Blood ALDH CD+ CD34CD+CD+ CD Marker Mean STDEV Mean STDEV Mean STDEV CD3 1.95 1.249 3.15 1.86343.69 14.643 CD7 7.36 14.051 6.30 6.090 43.76 17.460 CD10 0.46 0.4101.07 1.334 0.79 0.847 CD13 16.33 21.495 14.60 21.476 22.12 10.275 CD141.13 1.359 0.25 0.188 22.62 3.440 CD19 0.55 0.660 1.53 0.872 6.45 3.990CD33 12.33 13.595 9.70 12.607 23.73 3.985 ALDH 81.41 5.252 74.40 11.4540.90 0.517 CD35 1.01 0.974 1.22 1.078 20.08 8.910 CD38 49.20 21.90047.80 22.845 71.88 18.735 CD41-C 16.89 19.567 16.51 21.023 22.33 5.725CD45-M 78.28 20.454 75.61 25.070 88.84 12.651 CD56-BD 4.64 9.899 3.177.043 5.90 7.961 CD90 8.00 14.188 6.14 11.781 6.24 10.195 CD105 0.820.686 0.23 0.187 1.44 2.131 CD117 45.96 26.423 45.36 25.923 0.67 0.500CD127 0.39 0.255 0.14 0.159 0.59 1.012 CD133 29.61 21.858 30.74 24.1610.32 0.255 CD135 1.09 1.428 0.34 0.401 0.34 0.463 CD138 0.11 0.09 0.37HLA-DR 65.79 20.136 64.26 22.024 31.27 6.338 GLY-A 1.46 0.479 0.85 0.5563.07 2.110

TABLE 4C Summary of Peripheral Blood (All Gates) Peripheral Blood ALDHCD+ CD34CD+ CD+ CD Marker Mean STDEV Mean STDEV Mean STDEV CD3 1.471.426 1.86 2.371 21.81 18.080 CD7 1.02 0.845 1.29 0.976 14.85 15.860CD10 0.47 0.721 0.61 0.737 0.72 0.933 CD13 24.84 26.139 25.16 25.96245.08 27.214 CD14 0.66 0.798 0.57 0.556 25.43 11.399 CD19 0.60 0.7620.67 0.837 4.69 8.967 CD33 18.71 22.23 18.25 22.459 25.98 17.523 ALDH94.85 4.405 83.41 11.946 0.98 1.082 CD35 2.37 2.65 2.06 2.040 41.6721.568 CD38 69.17 26.779 67.65 27.161 36.31 18.805 CD41-C 27.26 23.16626.94 23.251 41.81 19.781 CD45-M 86.08 21.323 84.27 23.342 96.93 4.692CD56-BD 1.62 2.269 1.51 2.186 3.98 4.527 CD90 2.52 2.356 1.75 1.868 0.720.347 CD105 1.03 1.188 0.78 1.128 2.36 4.736 CD117 18.99 18.045 17.9318.403 0.24 0.308 CD127 0.54 0.657 0.40 0.489 1.03 2.686 CD133 36.8026.149 35.63 26.734 0.27 0.151 CD135 0.58 0.717 0.43 0.632 0.10 0.080CD138 0.57 0.582 0.43 0.584 0.04 0.012 HLA-DR 64.89 26.688 62.44 26.42721.49 10.799 GLY-A 0.69 0.694 0.51 0.611 0.47 0.379 ALDH 1.11 1.084 1.111.085 1.11 1.085

Example 2 Visualization and Characterization of the ALDH^(br)SSC^(lo)Cells Having the Phenotype of Interest

For intracellular staining, FACS™ isolated ALDH^(br)SSC^(lo) cellshaving the phenotype of interest, as identified in Example 1, arestained using the Fix and Penn cell permeabilization kit according tothe manufacturer's instructions (Caltag Laboratories). In preparationfor differential Wright Giemsa staining, cells in PBS are pelleted for 3minutes at 1000 rpm directly onto coated slides using a Cytospin3centrifuge. The cells are stained with Wright Giemsa stain in anautomated cell stainer.

Example 3 Functional Characterization of ALDH^(br)SSC^(lo), Cells Havingthe Phenotype of Interest Using In Vitro Culture

Hematopoietic progenitor colony HPC assays are performed by plating100-200 ALDH^(br)SSC^(lo) cells having the phenotype of interest, asidentified in Example 1, in MethoCult H4431 (StemCell Technologies,Inc.). The cells are incubated in a humidified chamber at 37° C. with 5%CO₂. Hematopoietic colonies (>100 cells) are then scored at 14-18 daysafter initiating the cultures. See, for example, Fallon et al (2003) Br.J. Haematol. 121:1, herein incorporated by reference in its entirety.

Long-term culture (LTC) assays are performed by maintainingALDH^(br)SSC^(lo) cells having the phenotype of interest, as identifiedin Example 1, on either irradiated allogeneic bone marrow stroma orstromal layers of murine MS-5 cells (Issaad et al. (1993) Blood 81:2916)or in some instances S17 cells (with medium containing Flt3 ligand,IL-3, erythropoietin, G-CSF, and IL-15) according to the method ofFallon et al. (2003) Br. J. Haematol. 121:1. MS-5 cells support thegrowth of human multipotent cells and BFU-E for extended periods of timeand may support early stem cells as well as, or better than, standardallogeneic stroma. The stromal layers are established by seeding thecenter wells of 24-well plates (Corning Costar Corp.) with 6-7×10⁴ MS-5cells/well in 0.5 ml DMEM supplemented with 10% FCS. These cells arecultured at 37° C. in a humidified incubator until the cultures approachapproximately 80% confluence. The monolayers are then irradiated with 30Gy γ-irradiation from a cesium source. After irradiation, the culturemedia from the monolayers is replaced entirely with Myelocult H5100(StemCell Technologies, Inc.) and the cells are maintained at 33° C. ina humidified chamber with 5% CO₂. Long-term cultures are typicallyinitiated with 500-20,000 sorted hematopoietic progenitor cells/well onthe irradiated MS-5 cells. At 7-10 day intervals, half the media fromeach well is removed so that the media can be replenished. Adherent andnon-adherent cells are harvested after 2 weeks and plated into HPCassays as described above. The HPC assay generally identifies relativelymature progenitor cells with limited lineage and self-renewal potential,while the LTC assay generally quantifies more primitive cells with ahigher self-renewal potential.

Example 4 Large Segmental Canine Femoral Defects are Healed withAutologous Stem Cell Therapy

The stem cells of the invention are used in a canine model of boneengraftment. See, for example, U.S. Pat. No. 6,541,024 hereinincorporated by reference in its entirety. The following protocols areused for culture-expanding autologous mesenchymal stem cells of theinvention that can regenerate clinically significant bone defects in alarge animal model.

MSC Cultivation and Manipulation

A 15 cc bone marrow aspirate is obtained from the iliac crest of eachanimal, according to an IACUC-approved protocol, and shipped on ice byovernight courier to the cell culture facilities. Isolation of canineALDH^(br) MSCs is achieved using procedures described in Example 1.Tissue culture flasks (185 cm²) are seeded with 10⁷ nucleated cellsisolated from the cushion, and cultured with DMEM containing 10% fetalcalf serum from a selected lot. Cells are passaged at 8×10³ cells/cm²,and maintained until the time of implantation. Cell-loaded implants areprepared by incubating fibronectin-coated poroushydroxyapatite-tricalcium phosphate (HA/TCP) cylinders (Zimmer, Inc.) ina 7.5×10⁶ cells/ml suspension of MSCs for 3 hr at 37° C. The intervalbetween marrow harvest and implantation is 16 days. An aliquot of cellsfrom each preparation is also cultured under osteoinductive conditionsto quantify aspects of osteoblastic differentiation.

Canine Femoral Gap Model

A unilateral segmental femoral defect model is developed for this studyfollowing IACUC approval. Under general anesthesia, thirty-sixskeletally mature female purpose-bred hounds (20 kg) undergo resectionof a 21 mm long osteoperiosteal segment from their mid-diaphysis. A 4.5mm Synthes™ 8-hole lengthening plate is contoured to the lateral aspectof the bone, and secured with bicortical screws. The defect is filledwith one of three materials; 1) a cell-free HA/TCP cylinder, 2) anMSC-loaded HA/TCP cylinder, or 3) cancellous bone harvested from theiliac crest. HA/TCP implants are secured by placing two sutures aroundthe implant and the plate. Animals receive peri-operative antibiotics,and analgesics are administered for three days post-operatively.

Radiographic and Histologic Analyses

Standard radiographic images are obtained at pre-op, immediatelypost-op, and at 4 week intervals until termination of the study. Allsamples contain a radiodensity step wedge to provide a basis forcomparing changes over time, and between dogs. Upon sacrifice, specimensare subjected to high resolution Faxitron radiography, and subsequentlyprocessed for biomechanical evaluation. Following torsion testing,undecalcified longitudinal sections will be processed for quantitativehistomorphometry.

Biomechanical Testing

Sixteen weeks after implantation, animals are sacrificed for torsiontesting of femurs. The fixation plate, screws, and adherent soft tissueare removed, and the metaphyses of the bones are embedded. The specimensare externally rotated in a custom torsion test apparatus, failure loadand stiffness recorded, and the data analyzed by one way ANOVA accordingto post hoc Student-Newman-Keuls tests.

Results

Generally, animals tolerate the surgical procedure well, with noincidence of infection, implant rejection, or failure of fixation. Twomodes of repair are generally observable in the MSC-loaded samples;first, considerable callus formation generally occurs at bothhost-implant interfaces; and second, a substantial collar of bonesurrounding the implant itself develops. Cell-free implants generally donot possess either of these features. Autograft samples generallyundergo a traditional consolidation sequence, with the majority of bonebeing laid down in the medial aspect of the gap defect. Generally,MSC-loaded samples not only became fully integrated at the host implantinterface, but the periosteal collar extended proximally and distallybeyond the cut edges of the gap. Furthermore, the diameter of new boneat the mid-diaphysis is generally greater in MSC-loaded implants thaneither autograft samples or intact limbs. Analyses of the osteogenicpotential MSCs from each animal generally demonstrate the development ofalkaline phosphatase-positive cells which deposit significantmineralized extracellular matrix.

Example 5 Myocardial Infarcts are Healed with Autologous Stem CellTherapy

The stem cells of the invention are used in a porcine model ofmyocardial infarction. See for example, Dib et al (2002) J. Endovasc.Ther. 9:313. The following protocols are used for culture-expandingautologous mesenchymal stem cells of the invention that can regenerateclinically significant heart damage in a large animal model.

Animal Preparation

7-month-old swine undergo baseline cardiovascular evaluation and serveas the recipient animal for myoblast transplantation. A 12-leadelectrocardiogram (ECG) is obtained, and 2-dimensional transthoracicechocardiography is performed to assess wall motion and ejectionfraction. An 8-F arterial sheath is inserted into the right femoralartery using a cutdown technique, and selective left and right coronaryangiography and left ventriculography are performed using the rightanterior oblique and left anterior oblique projections.

Three-dimensional (3D) electromechanical mapping is performed using theNOGA Biosense Navigational System via a 7-F NOGA B-curve catheteradvanced through the 8-F sheath into the left ventricle. Theelectromechanical activity of the myocardium is determined based on themaps constructed from 35 acquired points.

Concurrent with the femoral cutdown, a 6-cm incision is madelongitudinally along the right hind leg. Under sterile conditions, a 5-gsegment of the thigh muscle is removed. The muscle segment is placed ina cell transportation medium on ice and sent to a cell culturingfacility for myoblast expansion.

Infarction Model

An anterior infarct is induced in the host swine via coil embolizationusing individual 3- and 4-mm Vortx™ coils delivered to the middle leftanterior descending artery. Coronary occlusion occurs 2 minutes aftercoil placement, as demonstrated by coronary angiography. Apostinfraction left ventriculogram, echo cardiogram, and ECG areperformed within 5 minutes of infarction. Significant ventriculararrhythmias are treated with a 2% intravenous lidocaine bolus andelectrical cardioversion. The femoral artery is sutured, the incision isclosed, and the animal was is per standard operating procedures

Myoblast Isolation

The skeletal muscle myoblasts are isolated through a series of stepsdescribed in Example 1. The cells are plated in a growth medium composedof Dulbecco's Modified Eagle Medium with 10% (vol/vol) fetal bovineserum and 0.5% (vol/vol) gentamicin. The cells are maintained at <70%density and allowed to expand over a 4-week period, replacing one halfof the growth medium with fresh medium every other day. Cells arevisually examined daily and growth curves are obtained.

In preparation for cell injection, the myoblasts are harvested using0.05% trypsin/ethylenediaminetetraacetic acid; trypsin is inactivated bythe addition of growth medium containing fetal bovine serum. The cellsare centrifuged and washed twice. The resulting cell pellet isresuspended in 2.5 mL of serum-free Dulbecco's Modified Eagle Medium.

Allogenic Myoblast Transplantation

Four weeks after creating the infarction, the animals are anesthetizedwith intramuscular Telozol (tiletamine hydrochloride and zolazepamhydrochloride; 500 mg), intubated, and mechanically ventilated with 2%isoflurane and 3-L/min oxygen. An 8-F arterial sheath is inserted intothe left femoral artery using a cutdown technique and cardiovascularassessments are repeated (ECG, echocardiography, left ventriculography,and coronary angiography). Electromechanical mapping identifies theinfracted area.

Using a B-curve needle injection catheter calibrated to extend 4 to 5 mminto the endocardium, approximately 200 million cells in the 2.5-mLsuspension are injected into 25 arbitrarily selected sites (0.1 mL each)at the center and periphery of the infracted myocardium. Penetration ofthe endocardium is verified by ST elevations and premature ventricularcontractions during needle advancement. The injection sites aredelineated on the electromechanical map. After the injections arecompleted, the sheath is removed, the femoral artery is sutured, and theanimal is allowed to recover.

Ten days later, the animals are anesthetized and euthanized. The heartis harvested, rinsed in saline, and preserved in 10% formalin prior tosection in 5-mm increments from apex to base. Thin sections (8 μm) fromthe infarct region are stained either with hematoxylin and eosin (H&E)or Masson's trichrome.

Generally after the above procedures, cardiac function is restored topre-infarction levels. This includes functional measures both foroverall cardiac function via ECG and histological function as measuredby pathology performed on tissues after animal sacrifice.

Example 6 Characterization of Bone Marrow Derived Cells in Culture

Bone marrow (Sample #40411) was processed as in Example 1. Phenotype wasdetermined on the starting population. The ALDH^(br)SSC^(lo) populationwas sorted, and 1.8×10⁵ cells were cultured in CambrexMSCGM™-Mesenchymal Stem Cell Medium (Product #3238). Non-adherent cellswere removed after 7 days, and adherent cells were expanded followingmanufacturer's instructions for 4 weeks when the phenotype panel wasrepeated. Results show the percentage of cells expressing ALDH and othermarkers before and after the culture period (Table 5). These dataillustrate that adherent cells with the phenotype of mesenchymal stemcells grew out.

TABLE 5 % Cells Expressing marker at time shown Marker(s) Start Week 4ALDH^(br)SSC^(lo) 0.6 10.9 CD34 0.9 0.3 CD133 0.2 0.2 CD34⁺CD133⁺ 13.90.0 CD73 96.7 CD105 0.6 95.2 CD166 72.8 VEGF-R2 0.5 CD45 0.9 CD31 0.9ALDH^(br)SSC^(lo)/CD34⁺ 80.9 0.0 ALDH^(br)SSC^(lo)/CD133⁺ 21.6 0.0ALDH^(br)SSC^(lo)/CD73⁺ 97.3 ALDH^(br)SSC^(lo)/CD105⁺ 23.7 95.7ALDH^(br)SSC^(lo)/CD166⁺ 66.4 ALDH^(br)SSC^(lo)/VEGF-R2⁺ 0.2ALDH^(br)SSC^(lo)/CD45⁻ 99.5 ALDH^(br)SSC^(lo)/CD31⁺ 0.3ALDH^(br)SSC^(lo)/CD14⁺ 0.4 CD14 1.4

Example 7 Characterization and Culture of Bone Marrow Derived Cells fromMultiple Donors

Hematopoietic progenitor colony assays were performed forALDH^(br)SSC^(lo) cells sorted from normal human bone marrow. Bonemarrow samples with phenotypes shown on Table 6 were sorted to prepareALDH^(br)SSC^(lo) populations.

TABLE 6 % of cells with marker phenotype shown in marrow from donor #shown Marker(s) 30411 30418 30426 40404 Average ALDH^(br)SSC^(lo) 0.61.4 1.5 1.2 1.2 CD34 0.9 1.1 1.8 0.9 1.2 CD133 0.2 0.1 0.0 0.0 0.1CD34⁺CD133⁺ 13.9 1.5 1.2 0.0 4.1 CD73 3.3 1.1 2.2 CD105 0.6 1.0 1.4 0.20.8 CD166 0.7 0.1 0.4 VEGF-R2 0.4 8.8 0.2 3.1 CD45 95.2 92.8 94.0 CD3178.4 70.8 74.6 ALDH^(br)SSC^(lo)/CD34⁺ 80.9 37.1 30.7 27.3 44.0ALDH^(br)SSC^(lo)/CD133⁺ 21.6 7.2 1.0 0.2 7.5 ALDH^(br)SSC^(lo)/CD73⁺0.9 0.4 0.7 ALDH^(br)SSC^(lo)/CD105⁺ 23.7 46.9 52.8 5.2 32.1ALDH^(br)SSC^(lo)/CD166⁺ 5.8 0.3 3.0 ALDH^(br)SSC^(lo)/VEGF-R2⁺ 11.5 9.30.6 7.2 ALDH^(br)SSC^(lo)/CD45⁻ 36.7 50.1 43.4 ALDH^(br)SSC^(lo)/CD31⁺38.6 33.2 35.9

Cells were cultured for two weeks in standard methylcellulose colonyforming assays. All cultures formed multilineage colonies (FIG. 7). Datashown in FIG. 7 are total colony counts per 1000 cells input. Filledbars are data for marrow samples following removal of erythrocytes withammonium chloride treatment; open bars are cell suspensions followingreaction with the ALDH substrate but prior to sorting; and hatched barsare enriched ALDH^(br)SSC^(lo) populations after staining. These dataillustrate that markers typical of earlier endothelial progenitors suchas CD31 and VEGF receptor are present and, in most cases, enriched inthe ALDH^(br) population.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

1. A cell population of ALDH^(br) stem cells, wherein at least 10% ofthe cells within said population express at least CD105, and whereinsaid population is capable of multilineage development.
 2. The cellpopulation of claim 1, wherein said stem cells are derived from bonemarrow.
 3. The cell population of claim 1, wherein at least 10% of thecells within said population also express at least one cell surfacemarker selected from the group consisting of CD34, CD38, CD41, CD45,CD117, CD133, HLA-DR, and combinations thereof, wherein said populationis substantially free of cells expressing cell surface markers selectedfrom the group consisting of CD3, CD7, CD10, CD13, CD14, CD19, CD33,CD35, CD56, CD127, CD138, glycophorin A, and combinations thereof, andwherein said population of cells is capable of multilineage development.4. (canceled)
 5. The cell population of claim 1, wherein greater thanabout 60% of the cells within the population express the cell surfacemarker CD105.
 6. The cell population of claim 1, wherein at least 10% ofthe cells within the population are side scatter channel low (SSC^(lo)).7. (canceled)
 8. The cell population of claim 1, wherein said populationis capable of engrafting a mammal.
 9. The cell population of claim 8,wherein said population is capable of engrafting hematopoietic cells.10. The cell population of claim 8, wherein said population is capableof engrafting a SCID/NOD mouse spleen with human B cell precursors. 11.The cell population of claim 8, wherein said population is capable ofengrafting human thymus tissue transplanted into SCID/hu Thy mice with Tcell precursors.
 12. The cell population of claim 11, wherein said Tcell precursors are capable of developing into T cells expressing CD4 orCD8.
 13. The cell population of claim 8, wherein said population iscapable of engrafting mesenchymal cells.
 14. The cell population ofclaim 13, wherein said population is capable of engrafting tissueselected from the group consisting of bone marrow stroma, bone,cartilage, tendon, fat, smooth muscle, cardiac muscle, skeletal muscle,nerves, oligodendrocytes, fibroblasts, endothelium, and combinationsthereof.
 15. A composition comprising the cell population of claim 1 ina pharmacologically acceptable carrier.
 16. A method of reconstitutingblood tissue in a patient in need thereof, said method comprisingintroducing the cell population of claim 1 into said patient, whereinsaid cells are capable of engraftment.
 17. The method of claim 16,wherein said patient is in need of treatment for bone marrow ablation.18. The method of claim 16, wherein said patient is in need of treatmentfor cancer.
 19. The method of claim 16, wherein said patient is in needof treatment for sequelae related to cancer therapy.
 20. The method ofclaim 16, wherein at least 10% of the cells within said populationexpress a cell surface marker selected from the group consisting ofCD34, CD38, CD41, CD45, CD117, CD133, HLA-DR, and combinations thereof,wherein said population is substantially free of cells expressing cellsurface markers selected from the group consisting of CD3, CD7, CD10,CD13, CD14, CD19, CD33, CD35 CD56, CD127 CD138, glycophorin A, andcombinations thereof, and wherein said population of cells is capable ofmultilineage development.
 21. (canceled)
 22. The method of claim 16,wherein at least 10% of the cells within said population are sidescatter channel low (SSC^(lo)).
 23. (canceled)
 24. A method of repairingor regenerating a mesenchymal tissue in a patient in need thereof, saidmethod comprising introducing the cell population of claim 1 into saidpatient.
 25. The method of claim 24, wherein said mesenchymal tissue isselected from the group consisting of bone, cartilage, fat, endothelium,muscle, and combinations thereof.
 26. The method of claim 25, whereinsaid cell population of claim 1 promotes neovascularization.
 27. Themethod of claim 24, wherein said population is introduced to correct abone defect.
 28. The method of claim 24, wherein said population isintroduced to correct a cartilage defect.
 29. The method of claim 24,wherein at least 10% of the cells within said population express a cellsurface marker selected from the group consisting of CD34, CD38, CD41,CD45, CD117, CD133, HLA-DR, and combinations thereof, wherein saidpopulation is substantially free of cells expressing cell surfacemarkers selected from the group consisting of CD3, CD7, CD10, CD13,CD14, CD19, CD33, CD35, CD56, CD127, CD138, glycophorin A, andcombinations thereof, and wherein said population of cells is capable ofmultilineage development.
 30. (canceled)
 31. The method of claim 24,wherein at least 10% of the cells within said population are sidescatter channel low (SSC^(lo)).
 32. (canceled)
 33. A method of inducingimmunological tolerance in a patient in need thereof, said methodcomprising introducing said cell population of claim 1 into saidpatient, wherein said cells are capable of downregulating alloantigenrecognition and response.
 34. The method of claim 33, wherein saidpopulation is introduced to prevent graft versus host disease.
 35. Themethod of claim 33, wherein said population is introduced to ameliorategraft versus host disease.
 36. The method of claim 33, wherein at least10% of the cells within said population express a cell surface markerselected from the group consisting of CD34, CD38, CD41, CD45, CD117,CD133, HLA-DR, and combinations thereof, wherein said population issubstantially free of cells expressing cell surface markers selectedfrom the group consisting of CD3, CD7, CD10 CD13, CD14, CD19, CD33, CD35CD56 CD127 CD138, glycophorin A, and combinations thereof and whereinsaid population of cells is capable of multilineage development. 37.(canceled)
 38. The method of claim 33, wherein at least 10% of the cellswithin said population are side scatter channel low (SSC^(lo)). 39.(canceled)
 40. A method of producing neurons or oligodendrocytes in apatient in need thereof, said method comprising introducing the cellpopulation of claim 1 into said patient, wherein said cells are capableof differentiating into nervous tissue.
 41. The method of claim 40,wherein said population is introduced to prevent neural degeneration.42. The method of claim 40, wherein said population is introduced toameliorate neural damage or degeneration.
 43. The method of claim 40,wherein at least 10% of the cells within said population express a cellsurface marker selected from the group consisting of CD34, CD38, CD41,CD45, CD117, CD133, HLA-DR, and combinations thereof, wherein saidpopulation is substantially free of cells expressing cell surfacemarkers selected from the group consisting of CD3, CD7, CD10, CD13,CD14, CD19, CD33, CD35, CD56, CD127, CD138, glycophorin A, andcombinations thereof, and wherein said population of cells is capable ofmultilineage development.
 44. (canceled)
 45. The method of claim 40,wherein at least 10% of the cells within said population are sidescatter channel low (SSC^(lo)).
 46. (canceled)
 47. A method of producingcardiomyocytes in a patient in need thereof, said method comprisingintroducing the cell population of claim 1 into said patient, whereinsaid cells are capable of differentiating into heart tissue.
 48. Themethod of claim 47, wherein said population is introduced to preventischemic heart injury.
 49. The method of claim 47, wherein saidpopulation is introduced to ameliorate ischemic heart injury.
 50. Themethod of claim 47, wherein at least 10% of the cells within saidpopulation express a cell surface marker selected from the groupconsisting of CD34, CD38, CD41, CD45, CD117, CD133, HLA-DR, andcombinations thereof, wherein said population is substantially free ofcells expressing cell surface markers selected from the group consistingof CD3, CD7, CD10, CD13, CD14, CD19, CD33, CD35, CD56, CD127, CD138,glycophorin A, and combinations thereof, and wherein said population ofcells is capable of multilineage development.
 51. (canceled)
 52. Themethod of 47, wherein at least 10% of the cells within said populationare side scatter channel low (SSC^(lo)).
 53. (canceled)
 54. A cellpopulation of bone-marrow-derived, ALDH^(br) stem cells, wherein saidcells are capable of multilineage development.
 55. The cell populationof claim 54, wherein at least 10% of the cells within said populationexpress a cell surface marker selected from the group consisting ofCD34, CD38, CD41, CD45, CD105, CD117, CD133, HLA-DR, and combinationsthereof, wherein said population is substantially free of cellsexpressing cell surface markers selected from the group consisting ofCD3, CD7, CD10 CD13, CD14, CD19, CD33, CD35, CD56, CD127, CD138,glycophorin A, and combinations thereof, and wherein said population ofcells is capable of multilineage development.
 56. The cell population ofclaim 55, wherein at least 10% of the cells within said populationexpress at least CD105.
 57. The cell population of claim 56, wherein atleast 40% of the cells within said population express at least CD105.58. (canceled)
 59. The cell population of claim 54, wherein at least 10%of the cells within the population are side scatter channel low(SSC^(lo)).
 60. (canceled)
 61. The cell population of claim 54, whereinsaid population is capable of engrafting a mammal.
 62. The cellpopulation of claim 61, wherein said population is capable of engraftinghematopoietic cells.
 63. The cell population of claim 61, wherein saidpopulation is capable of engrafting a SCID/NOD mouse spleen with human Bcell precursors.
 64. The cell population of claim 61, wherein saidpopulation is capable of engrafting human thymus tissue transplantedinto SCID/hu Thy mice with T cell precursors.
 65. The cell population ofclaim 64, wherein said T cell precursors are capable of developing intoT cells expressing CD4 or CD8.
 66. The cell population of claim 61,wherein said population is capable of engrafting mesenchymal cells. 67.The cell population of claim 66, wherein said population is capable ofengrafting tissue selected from the group consisting of bone marrowstroma, bone, cartilage, tendon, fat, smooth muscle, cardiac muscle,skeletal muscle, nerves, oligodendrocytes, fibroblasts, endothelium, andcombinations thereof.
 68. A composition comprising the cell populationof claim 54 in a pharmacologically acceptable carrier.
 69. A method ofreconstituting blood tissue in a patient in need thereof, said methodcomprising introducing the cell population of claim 54 into saidpatient, wherein said cells are capable of engraftment.
 70. The methodof claim 69, wherein said patient is in need of treatment for bonemarrow ablation.
 71. The method of claim 69, wherein said patient is inneed of treatment for cancer.
 72. The method of claim 69, wherein saidpatient is in need of treatment for sequelae related to cancer therapy.73. The method of claim 69, wherein at least 10% of the cells withinsaid population express a cell surface marker selected from the groupconsisting of CD34, CD38, CD41, CD45, CD105, CD117, CD133, HLA-DR, andcombinations thereof, wherein said population is substantially free ofcells expressing cell surface markers selected from the group consistingof CD3, CD7, CD10, CD13, CD14, CD19, CD33, CD35, CD56, CD127, CD138,glycophorin A, and combinations thereof, and wherein said population ofcells is capable of multilineage development.
 74. The method of claim73, wherein at least 10% of the cells within said population express atleast CD105.
 75. The method of claim 73, wherein at least 40% of thecells within said population express at least CD105.
 76. (canceled) 77.The method of claim 69, wherein at least 10% of the cells within saidpopulation are side scatter channel low (SSC^(lo)).
 78. (canceled)
 79. Amethod of repairing or regenerating a mesenchymal tissue in a patient inneed thereof, said method comprising introducing the cell population ofclaim 54 into said patient.
 80. The method of claim 79, wherein saidmesenchymal tissue is selected from the group consisting of bone,cartilage, fat, endothelium, muscle, and combinations thereof.
 81. Themethod of claim 80, wherein said cell population promotesneovascularization.
 82. The method of claim 79, wherein said populationis introduced to correct a bone defect.
 83. The method of claim 79,wherein said population is introduced to correct a cartilage defect. 84.The method of claim 79, wherein at least 10% of the cells within saidpopulation express a cell surface marker selected from the groupconsisting of CD34, CD38, CD41, CD45, CD105, CD117, CD133, HLA-DR, andcombinations thereof, wherein said population is substantially free ofcells expressing cell surface markers selected from the group consistingof CD3, CD7, CD10, CD13, CD14, CD19, CD33, CD35, CD56, CD127, CD138,glycophorin A, and combinations thereof, and wherein said population ofcells is capable of multilineage development.
 85. The method of claim84, wherein at least 10% of the cells within said population express atleast CD105.
 86. The method of claim 84, wherein at least 40% of thecells within said population express at least CD105.
 87. (canceled) 88.The method of claim 79, wherein at least 10% of the cells within saidpopulation are side scatter channel low (SSC^(lo)).
 89. (canceled)
 90. Amethod of inducing immunological tolerance in a patient in need thereof,said method comprising introducing the cell population of claim 54 intosaid patient, wherein said cells are capable of downregulatingalloantigen recognition and response.
 91. The method of claim 90,wherein said population is introduced to prevent graft versus hostdisease.
 92. The method of claim 90, wherein said population isintroduced to ameliorate graft versus host disease.
 93. The method ofclaim 90, wherein at least 10% of the cells within said populationexpress a cell surface marker selected from the group consisting ofCD34, CD38, CD41, CD45, CD105, CD117, CD133, HLA-DR, and combinationsthereof, wherein said population is substantially free of cellsexpressing cell surface markers selected from the group consisting ofCD3, CD7, CD10, CD13, CD14, CD19, CD33, CD35, CD56, CD127, CD138,glycophorin A, and combinations thereof, and wherein said population ofcells is capable of multilineage development.
 94. The method of claim93, wherein at least 10% of the cells within said population express atleast CD105.
 95. The method of claim 93, wherein at least 40% of thecells within said population express at least CD105.
 96. (canceled) 97.The method of claim 90, wherein at least 10% of the cells within saidpopulation are side scatter channel low (SSC^(lo)).
 98. (canceled)
 99. Amethod of producing neurons or oligodendrocytes in a patient in needthereof, said method comprising introducing the cell population of claim54 into said patient, wherein said cells are capable of differentiatinginto nervous tissue.
 100. The method of claim 99, wherein saidpopulation is introduced to prevent neural degeneration.
 101. The methodof claim 99, wherein said population is introduced to ameliorate neuraldamage or degeneration.
 102. The method of claim 99, wherein at least10% of the cells within said population express a cell surface markerselected from the group consisting of CD34, CD38, CD41, CD45, CD105,CD117, CD133, HLA-DR, and combinations thereof, wherein said populationis substantially free of cells expressing cell surface markers selectedfrom the group consisting of CD3, CD7, CD10, CD13, CD14, CD19, CD33,CD35, CD56, CD127, CD138, glycophorin A, and combinations thereof, andwherein said population of cells is capable of multilineage development.103. The method of claim 102, wherein at least 10% of the cells withinsaid population express at least CD105.
 104. The method of claim 102,wherein at least 40% of the cells within said population express atleast CD105.
 105. (canceled)
 106. The method of claim 99, wherein atleast 10% of the cells within said population are side scatter channellow (SSC^(lo)).
 107. (canceled)
 108. A method of producingcardiomyocytes in a patient in need thereof, said method comprisingintroducing the cell population of claim 54 into said patient, whereinsaid cells are capable of differentiating into heart tissue.
 109. Themethod of claim 108, wherein said population is introduced to preventischemic heart injury.
 110. The method of claim 108, wherein saidpopulation is introduced to ameliorate ischemic heart injury.
 111. Themethod of claim 108, wherein at least 10% of the cells within saidpopulation express a cell surface marker selected from the groupconsisting of CD34, CD38, CD41, CD45, CD105, CD117, CD133, HLA-DR, andcombinations thereof, wherein said population is substantially free ofcells expressing cell surface markers selected from the group consistingof CD3, CD7, CD10, CD13, CD14, CD19, CD33, CD35, CD56, CD127, CD138,glycophorin A, and combinations thereof, and wherein said population ofcells is capable of multilineage development.
 112. The method of claim111, wherein at least 10% of the cells within said population express atleast CD105.
 113. The method of claim 111, wherein at least 40% of thecells within said population express at least CD105.
 114. (canceled)115. The method of claim 108, wherein at least 10% of the cells withinsaid population are side scatter channel low (SSC^(lo)).
 116. (canceled)117. A method of screening a compound for its ability to promotedifferentiation, growth, cytoxicity, apoptosis, or engraftment of stemcells, comprising the steps of: a) isolating ALDH^(br) stem cells from astem cell source; b) further selecting a subpopulation of cellsexpressing CD105; and c) contacting said subpopulation of cells withsaid compound.
 118. A method of screening a compound for its ability topromote differentiation, growth, cytoxicity, apoptosis, or engraftmentof stem cells, comprising the steps of: a) isolating ALDH^(br) stemcells from bone marrow; b) further selecting a subpopulation ofALDH^(br) stem cells expressing markers selected from the groupconsisting of CD34, CD38, CD41, CD45, CD105, CD117, CD133, HLA-DR, andcombinations thereof; and c) contacting said subpopulation of ALDH^(br)cells with said compound.
 119. A kit comprising a detectable ALDHsubstrate disposed in a container, and antibodies specific for cellsurface markers selected from the group consisting of CD34, CD38, CD41,CD45, CD105, CD117, CD133, HLA-DR, and combinations thereof, disposed ina container.
 120. The kit of claim 119, wherein the ALDH substrate isBODIPY aminoacetaldehyde diethyl acetal or BODIPY aminoacetaldehyde.